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. 2021 Sep 3;10:e68542. doi: 10.7554/eLife.68542

Figure 5. Recombinant human diamine oxidase (rhDAO) heparin-binding motif (HBM) mutants are enzymatically fully active in vitro and in vivo.

(A) Purified rhDAO-R568S/R571T mutant rapidly and completely degrades histamine. 6 nM rhDAO-R568S/R571T with and without 100× high molecular weight heparin (HMWH) (600 nM) were incubated with 100 ng/mL histamine in 1% human serum albumin-phosphate buffered saline (HSA-PBS) and EDTA plasma of a healthy volunteer for 60 min at 37°C. Histamine concentrations were determined in duplicate. Plasma: △; plasma with 100× HMWH: ◇; HSA-PBS: ○; HSA-PBS with 100× HMWH: □. (B–D) rhDAO HBM mutants are enzymatically active in rats. Means of the measured values ± standard error of the mean (SEM) using 1 mg/kg rhDAO-R568S/R571T (B, n = 3; R2 = 94.8% with p-value<0.001, linear regression) and rhFcDAO-R568S/R571T (D, n = 3; R2 = 98% with p-value<0.001, linear regression) and two individually plotted measurements of rhDAO-R568S/K575T (C); rhDAO concentration (ng/mL; □ and ○) and activity (relative fluorescence units [RFU]; ■ and ●); RFU data downscaled by a factor 50 in panels (B) and (C) and by 100 in (D) for easier presentation. n: number of animals.

Figure 5.

Figure 5—figure supplement 1. Recombinant human diamine oxidase (rhDAO) antigen concentrations and activity are highly correlated in rat plasma.

Figure 5—figure supplement 1.

Means of the measured values using 1 mg/kg rhDAO-R568S/R571T (A, n = 3) and Fc-rhDAO-R568S/R571T (C, n = 3) and two individually plotted measurements of rhDAO-R568S/K575T (B); relative fluorescence units (RFU) data downscaled by factor 50 in panels (A) and (B) and by factor 100 in (C). n: number of animals.
Figure 5—figure supplement 2. The enzymatic activity of rhDAO-R568S/R571T is comparable to rhDAO-WT.

Figure 5—figure supplement 2.

Enzymatic activity of rhDAO-WT (□) and rhDAO-R568S/R571T (●) at 0.7 nM, 2 nM, and 6 nM was measured as published (Boehm et al., 2020; Boehm et al., 2017). Means ± standard deviation (SD) of duplicate measurements are shown. RLU: relative light units; rhDAO: recombinant human diamine oxidase.